There is a growing interest in using internal ribosome entry site (IRES) elements for expressing foreign genes in eukaryotic cells. Although the number of nucleotide sequences that are capable to provide cap-independent translation increases constantly, identification of new IRESes has been occasional or accidental without distinct methodology of predicting IRES elements.
In most cases, the regulation of translation in eukaryotic cells occurs at the stage of initiation by a scanning mechanism. This involves binding of eukaryotic initiation factor 4F (eIF4F), a complex of proteins which includes eIF4E (the cap binding protein), eIF4G (a large protein which acts as a scaffold for the proteins in the complex and has binding sites for eIF4E, eIF4a, eIF3, and a poly(A) binding protein), and eIF4A (RNA helicase) to the m7GpppN cap structure, recruitment of a 40 S ribosomal subunit, and scanning to the first AUG codon in the correct context (Pain, V. M., 1996, Eur. J. Biochem. 236, 747-771).
Translation initiation can also occur by a mechanism that does not require the cap structure. In this case, the ribosome enters the mRNA at a region termed an internal ribosome entry site (IRES) that is up to 1,000 bp long. IRESes were originally identified in picornaviral RNAs, and during picornaviral infection, there is often a switch of translation from host encoded cellular mRNAs to viral transcripts (Jackson and Kaminski, 1995, RNA 1, 985-1000).
IRES-containing animal mRNAs can presumably recruit 40 S ribosomes either via their capped 5′ ends or their IRES elements, which likely allows translation under conditions when cap-dependent translation is reduced as, for example, during viral infection, at the G2/M phase of cell cycle, apoptosis or stress conditions (Johannes et al., (1999) Proc. Natl. Acad. Sci. USA 96, 13118-13123; Cornelis et al., (2000) Molecular Cell 5, 597-605; Pyronnet et al., (2000) Molecular Cell 5, 607-616; Stein et al., 1998 Mol. and Cell. Biol. 18, 3112-3119; Holcik et al., 2000 Oncogene 19, 4174-4177; Stoneley et al., 2000 Mol. and Cell. Biol. 20, 1162-1169). Up to 3% of animal cellular mRNAs are translated despite that eIF4F-RNA binding is inhibited (Johannes et al., 1999).
In contrast to animal cellular mRNA, there are no published reports concerning IRES-mediated initiation of translation in plant cells in vivo. However, the uncapped 5′ leaders of several plant viral genomic RNAs including poty-, como-, and luteoviruses are responsible for cap-independent translation (Carrington and Freed, 1990; Gallie et al., 1995; Niepel and Gallie, 1999; Tacke et al., 1990; Thomas et al., 1991). Tobamoviruses and potexvirus X are the only examples of IRESes located in internal parts of viral genomes (Hefferon et al., 1997 J. Gen. Virol. 78, 3051-3059; Hefferon et al., 2000 Arch. Virol. 145, 945-956; Ivanov et al., (1997) Virology 232, 32-43; Skulachev et al., (1999) Virology 263, 139-154).
Tobacco mosaic tobamovirus (TMV) is a positive-stranded RNA plant virus with a monopartite genome of 6395 nucleotides (nt) in length (Goelet et al., 1982 Proc. Natl. Acad. Sci. USA 79, 5818-5822). The 5′ proximal ORFs encoding replicative proteins are expressed directly from the genomic RNA and the level of synthesis of the smaller (126 kDa) protein is approximately 10 times higher than the level of the 183 kDa protein which is produced by occasional readthrough of the stop codon for the 126-kDa ORF (Siegel et al., 1976 Virology 73, 363-371). Although some replication can occur with the larger protein only, both proteins are required for efficient replication (Ishikawa et al., 1986). The remaining TMV gene products, the movement protein (MP) and the coat protein (CP), are expressed from 3′ coterminal subgenomic mRNAs (sgRNAs) (reviewed by Palukaitis and Zaitlin, 1986 In: “The plant virus”. M. H. V. van Regenmortel and M. Fraenkel-Conrat, Eds. Vol. 2, pp. 105-131. Plenum Press, NY). Thus, the internal movement protein (MP) gene and the 3′-proximal coat protein gene cannot be translated from genomic RNA of typical tobamoviruses (TMV UI is the type member of the genus Tobamovirus). The dicistronic intermediate-length RNA-2 called sgRNA I2 RNA is translated to produce the 30-kDa MP (Bruening et al., 1976 Virology 71, 498-517; Higgins et al., 1976 Virology 71, 486-497; Beachy and Zaitlin, 1977 Virology 81, 160-169; Goelet and Karn, 1982 J. Mol. Biol. 154, 541-550), whereas the 3% proximal coat protein (CP) gene of I2 RNA is translationally silent. This gene is expressed only from the small monocistronic sgRNA (Beachy and Zaitlin, 1977).
It has been shown (Ivanov et al., (1997) Virology 232, 32-43) that, unlike the typical tobamoviruses, the translation of the CP gene of a crucifer-infecting tobamovirus (crTMV) occurs in vitro by an internal ribosome entry mechanism. The genome of crTMV (6312 nts) contains four traditional genes encoding two components of the replicase (proteins of 122-kDa and 178-kDa, the readthrough product of the 122-kDa protein), a 30-kDa MP and a 17-kDa CP (Dorokhov et al., 1993 Dokl. Russian Acad. Sci. 332, 518-52; Dorokhov et al., 1994 FEBS Lett. 350, 5-8). It was found that the 148-nt region upstream of the CP gene of crTMV RNA contains an internal ribosome entry site (IRESCP148CR) promoting the internal translation initiation of the CP gene and different reporter genes (Ivanov et al., 1997). By analogy with crTMV, the 3′-proximal CP gene of potato virus X occurs by a mechanism of internal initiation (Hefferon et al., 1997). The capacity of crTMV IRESCRCP for mediating internal initiation of translation distinguishes this tobamovirus from the well-known type member of the genus, TMV U1. The equivalent 148-nt sequence from TMV U1 RNA was incapable (UICP,148SP) of mediating internal initiation of translation (Ivanov et al., 1997).
Recently, it has been shown that the 228- and 75-nt regions upstream of the MP gene of crTMV and TMV U1 RNAs contain IRES elements, IRESMP,75CR and IRESMP,228CR, respectively, which direct expression of 3′-proximal reporter genes from dicistronic constructs in cell-free translation systems and in isolated protoplasts (Skulachev et al., 1999). Moreover, the equivalent sequence from TMV U1 RNA used as the intercistronic spacer (IRESMP,75U1) was able to mediate translation of the second gene in dicistronic transcripts.
Recent studies on plant viruses also gave new examples of cap-independent initiation of translation without IRES. RNAs of viruses and satellite viruses in the Luteovirus and Necrovirus genera, and the large Tombusviridae family lack both a 5′ cap and a 3′ poly(A) tail. However, they can be translated efficiently owing to different translation enhancement sequences located in the coding region close to their 3′ UTR. These sequences are different from IRES elements in two fundamental ways: they do not confer internal ribosome entry and they are located in the 3′ untranslated region (3′UTR). The structure and putative mechanism of action of these sequences are described in details for Barley Yellow Dwarf Virus (BYDV) (Guo et al., 2000 RNA 6, 1808-1820).
IRESes have been identified within some cellular and viral mRNAs of higher eukaryotes (mammals and plants), however, it remains unclear if IRESes do also exist within the mRNAs of lower eukaryotes. Recently, the 5′ leader sequences of two yeast mRNAs functioning as IRESs were identified (Zhou et al., 2001 Proc. Natl. Acad. Sci. USA 98, 1531-1536).
The sequences of the animal IRESes characterized are dissimilar (Jackson, 2000, see also FIG. 3) but, in one case, there is evidence of an important role of short nucleotide sequences in IRES functionality of Gtx homeodomain protein. The Gtx IRES contains several non-overlapping segments displaying complementarity to 18 S rRNA that were shown to mediate internal initiation (Hu et al., 1999. Proc. Natl. Acad. Sci. USA 96, 1339-1344). Within one of these segments, a 9-nt GC-rich sequence CCGGCGGGU which is 100% complementary to 18 S rRNA at nucleotides 1132-1124, was identified, and it was shown that synthetic IRESes (FIG. 4) composed of multiple linked copies of this 9-nt IRES module, support internal initiation in animal cells at a very high level (Chappel et al., 2000 Proc. Natl. Acad. Sci. USA 97, 1536-1541).
The low activity of animal viral IRESes (encephalomyocarditis virus IRES, IRESEMCV) in plants was reported (Urwin et al., 2000 Plant J. 24, 583-589). So far, there is no evidence for cross-kingdom (plant, animal, yeast) activity of any IRES element. Although the number of nucleotide sequences that are capable of providing cap-independent translation increases constantly, identification of new IRESes has been occasional and accidential, without methodology of prediction. Moreover, the known IRESes do not allow a fine adjustment of efficiency. They are taxonomically limited and structurally highly disparate.
Therefore, it is an object of the invention to provide a method of creating artificial IRES elements.
It is another object to provide artificial IRES elements having cross-kingdom activity.
It is another object of the invention to provide a method of creating IRES elements of a desired degree of efficiency.
It is a further object to provide a process of expressing a nucleotide sequence of interest in eukaryotic cells under translational control of a novel IRES element of the invention.
It is another object to provide a method of identifying nucleic acid elements having IRES activity by searching nucleotide sequences e.g. of data bases.